The project explores the mechanism of active transport of neurotransmitters through cytoplasmic membranes as well as across membranes of subcellular storage organelles. Active transport of neurotransmitters plays an important role in the process of synaptic transmission, both in the termination of the signal (high affinity cytoplasmic transporter) and in the scavenging and storage of the neurotransmitter (subcellular storage organelles). Drugs which interact with these transport systems have profound pharmacological effects due to changes in the concentration of the neurotransmitters. The purpose of the project is to characterize the transport systems both at a mechanistic and a molecular level. The studies will be in principle carried out with membrane vesicles prepared from cytoplasmic membranes derived from rat brain synaptosomes and from subcellular storage granules. The studies at the mechanistic level will be carried out based on the knowledge that ion gradients and membrane potentials play an important role in the transport process. The approach to be used includes measurement of transport in relation to the existing ion gradients, effects of ionophores and effects of artificially imposed gradients on transport, specificity of the system, unidirectional fluxes compared to net fluxes and binding measurements. The studies at the molecular level will include characterization of the proteins involved in the transport process through specific chemical labeling with appropriate markers and their isolation and purification. The various probes will be used in an effort to dissect the steps involved in the transfer of a solute, to study the architecture of the binding site, and the sidedness and assymetry of the transport protein. During different steps in the purification procedure we will assess biological activity of the protein by reconstitution through incorporation into single-walled artificial liposomes.